TY - JOUR
T1 - The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Gα-interaction face
AU - Kimple, Adam J.
AU - Willard, Francis S.
AU - Giguère, Patrick M.
AU - Johnston, Christopher A.
AU - Mocanu, Viorel
AU - Siderovski, David P.
N1 - Funding Information:
We thank Drs. Declan Doyle and Meera Soundararajan (SGC Oxford) for RGS protein expression vectors, and Dr. Dmitriy Gremyachinskiy (UNC) for his chemistry advice. A.J.K. gratefully acknowledges prior support of the UNC MD/PhD program (T32 GM008719) and current predoctoral support from National Institute of Mental Health (F30 MH074266). C.A.J. and D.P.S. were supported by National Institutes of Health grants F32 GM076944 and R03 NS053754, respectively.
PY - 2007/9
Y1 - 2007/9
N2 - Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and are thus crucial to the timing of G protein-coupled receptor (GPCR) signaling. Small molecule inhibition of RGS proteins is an attractive therapeutic approach to diseases involving dysregulated GPCR signaling. Methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986) was reported as a selective RGS4 inhibitor, but with an unknown mechanism of action [D.L. Roman, J.N. Talbot, R.A. Roof, R.K. Sunahara, J.R. Traynor, R.R. Neubig, Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay, Mol. Pharmacol. 71 (2007) 169-75]. Here, we describe its mechanism of action as covalent modification of RGS4. Mutant RGS4 proteins devoid of surface-exposed cysteine residues were characterized using surface plasmon resonance and FRET assays of Gα binding, as well as single-turnover GTP hydrolysis assays of RGS4 GAP activity, demonstrating that cysteine-132 within RGS4 is required for sensitivity to CCG-4986 inhibition. Sensitivity to CCG-4986 can be engendered within RGS8 by replacing the wildtype residue found in this position to cysteine. Mass spectrometry analysis identified a 153-Dalton fragment of CCG-4986 as being covalently attached to the surface-exposed cysteines of the RGS4 RGS domain. We conclude that the mechanism of action of the RGS protein inhibitor CCG-4986 is via covalent modification of Cys-132 of RGS4, likely causing steric hindrance with the all-helical domain of the Gα substrate.
AB - Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and are thus crucial to the timing of G protein-coupled receptor (GPCR) signaling. Small molecule inhibition of RGS proteins is an attractive therapeutic approach to diseases involving dysregulated GPCR signaling. Methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986) was reported as a selective RGS4 inhibitor, but with an unknown mechanism of action [D.L. Roman, J.N. Talbot, R.A. Roof, R.K. Sunahara, J.R. Traynor, R.R. Neubig, Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay, Mol. Pharmacol. 71 (2007) 169-75]. Here, we describe its mechanism of action as covalent modification of RGS4. Mutant RGS4 proteins devoid of surface-exposed cysteine residues were characterized using surface plasmon resonance and FRET assays of Gα binding, as well as single-turnover GTP hydrolysis assays of RGS4 GAP activity, demonstrating that cysteine-132 within RGS4 is required for sensitivity to CCG-4986 inhibition. Sensitivity to CCG-4986 can be engendered within RGS8 by replacing the wildtype residue found in this position to cysteine. Mass spectrometry analysis identified a 153-Dalton fragment of CCG-4986 as being covalently attached to the surface-exposed cysteines of the RGS4 RGS domain. We conclude that the mechanism of action of the RGS protein inhibitor CCG-4986 is via covalent modification of Cys-132 of RGS4, likely causing steric hindrance with the all-helical domain of the Gα substrate.
KW - CCG-4986
KW - RGS protein inhibitor
KW - RGS4
KW - Regulator of G-protein signaling
KW - Thiol adduct
UR - http://www.scopus.com/inward/record.url?scp=34548289765&partnerID=8YFLogxK
U2 - 10.1016/j.bbapap.2007.06.002
DO - 10.1016/j.bbapap.2007.06.002
M3 - Article
C2 - 17660054
AN - SCOPUS:34548289765
SN - 1570-9639
VL - 1774
SP - 1213
EP - 1220
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
IS - 9
ER -